Apparatus and method for assaying coagulation in fluid samples

ABSTRACT

This invention is a disposable cartridge for use at the patient side to perform traditional coagulation assays on fresh whole blood or blood derivative samples. The cartridge, in use with an electronic analyzer allows a fluid sample to be metered and quantitatively mixed with reagents which activate the coagulation cascade. An artificial substrate for thrombin, the enzyme whose action results in clot formation is also provided. Clot formation is subsequently detected using a microfabricated sensor also housed within the cartridge which detects electrochemically the product of the thrombin reaction upon the synthetic substrate.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority based upon my copendingProvisional Application Serial No. 60/164,935, filed Nov. 15, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates to an apparatus for conducting avariety of assays that are responsive to a change in the viscosity of asample fluid and relates to methods of conducting such assays. Inparticular, the present invention is related to the use of a cartridgefor conducting one or more coagulation assays. The present inventionmakes adventitious use of a pump means for moving a fluid sample. In oneembodiment, sample movement is achieved by reversibly, rapidly, andreproducibly applying pressure to a sample fluid to produce asubstantially reciprocating motion that is, in turn, detectable by anappropriate sensor. The disclosed device enjoys simplicity and isadaptable to the point-of-care clinical diagnostic area, including usein accident sites, emergency rooms or medical intensive care units.

BACKGROUND OF THE INVENTION

[0003] Keeping blood in a fluid state, termed hemostasis, requires asubtle balance of pro- and anticoagulants. Procoagulants preventexcessive bleeding by blocking blood flow from a damaged vessel, whereasanticoagulants prevent clots from forming in the circulating systemwhich could otherwise block blood vessels and lead to myocardialinfarction or stroke.

[0004] The biochemical sequence leading to a blood clot is termed thecoagulation cascade. The mechanism is based on catalytic conversion offibrinogen, a soluble plasma protein, to insoluble fibrin. The enzymecatalyzing this reaction is thrombin, which does not permanentlycirculate in the blood in an active form but exists as prothrombin, theinactive precursor of thrombin. Conversion to thrombin occurs in thepresence of calcium ions and tissue thromboplastin. This mechanism isknown as the extrinsic pathway. A second, more complex, intrinsicpathway is activated by clotting factors associated with platelets andis well understood in the art.

[0005] Diagnosis of hemorrhagic conditions such as hemophilia, where oneor more of the twelve blood clotting factors may be defective, can beachieved by a wide variety of coagulation tests. In addition, severaltests have been developed to monitor the progress of thrombolytictherapy. Other tests have been developed to signal a prethrombolytic orhypercoagulable state, or monitor the effect of administering protamineto patients during cardiopulmonary bypass surgery. However, the mainvalue of coagulation tests is in monitoring oral and intravenousanticoagulation therapy. Three of the key diagnostic tests are activatedpartial thromboplastin time (APTT), prothrombin time (PT), and activatedclotting time (ACT).

[0006] An APTT test evaluates the intrinsic and common pathways ofcoagulation. For this reason APTT is often used to monitor intravenousheparin anticoagulation therapy. Specifically, it measures the time fora fibrin clot to form after the activating agent, calcium, and aphospholipid have been added to the citrated blood sample. Heparinadministration has the effect of suppressing clot formation.

[0007] A PT test evaluates the extrinsic and common pathways ofcoagulation and, therefore, is used to monitor oral anticoagulationtherapy. The oral anticoagulant coumadin suppresses the formation ofprothrombin. Consequently, the test is based on the addition of calciumand tissue thromboplastin to the blood sample.

[0008] An ACT test evaluates the intrinsic and common pathways ofcoagulation. It is often used to monitor anticoagulation via heparintherapy. The ACT test is based on addition of an activator to theintrinsic pathway to fresh whole blood to which no exogenousanticoagulant has been added.

[0009] The standard laboratory technology for coagulation teststypically uses a turbidimetric method. For analysis, whole-blood samplesare collected into a citrate vacutainer and then centrifuged. The assayis performed with plasma to which a sufficient excess of calcium hasbeen added to neutralize the effect of citrate. For a PT test, tissuethromboplastin is provided as a dry reagent that is reconstituted beforeuse. This reagent is thermally sensitive and is maintained at 4 degreesC. by the instruments. Aliquots of sample and reagent are transferred toa cuvette heated at 37 degrees C., and the measurement is made based ona change in optical density.

[0010] As an alternative to the turbidimetric method, Beker et al. (See,Haemostasis (1982) 12:73) introduced a chromogenic PT reagent(Thromboquant PT). The assay is based on the hydrolysis ofp-nitroaniline from a modified peptide, Tos-Gly-Pro-Arg-pNA, by thrombinand is monitored spectrophotometrically.

[0011] Coagulation monitors are known for the analysis of whole blood.For example, a unit-use cartridge has been described in U.S. Pat. No.4,756,884 in which dry reagents are placed into the analyzer which isthen heated to 37 degrees C. before a drop of blood is introduced. Thesample is mixed with the reagent by capillary draw. The detectionmechanism is based on laser light passing through the sample. Bloodcells moving along the flow path- yield a speckled pattern specific tounclotted blood. When the blood clots, movement ceases producing apattern specific to clotted blood.

[0012] An automatic coagulation timer has been described which measuresthe activated clotting time (ACT) in blood samples from patients duringcardiopulmonary bypass. The sample is added to a cartridge whichincorporates a stirring device on to which the clot forms. Motion of thestirring device is controlled by a photo optical detector (See, Keeth etal., Proceedings Am. Acad. Cardiovascular Perfusion (1988) 9:22).

[0013] U.S. Pat. No. 4,304,853 discloses the use of a substrate whichproduces an electroactive product on reaction with the enzyme thrombin.A sensor is used to detect the electroactive product. The disclosuredoes not include a single-use cartridge and does not disclose the use ofa second sensor to monitor the location of the sample.

[0014] U.S. Pat. No. 4,497,744 discloses a turbidometric method forassaying coagulation. Plasma containing an excess of citrate is used inthe test. A reagent which induces clotting is added, the sample isplaced in a turbidometer, and coagulation is indicated by an increase inthe turbidity of the sample.

[0015] U.S. Pat. No. 5,096,669, incorporated herein by reference,includes the general format for use of a cartridge and analytzer forblood chemistry testing such as potassium and glucose blood levels andthe use of a pump to move a sample fluid to a sensor region in a singledirection.

[0016] U.S. Pat. No. 5,200,051, incorporated herein by reference,discloses efficient methods of microfabrication of sensor devices foranalysis of analytes.

[0017] U.S. Pat. No. 5,302,348 discloses a blood coagulation testapparatus in which blood is forced to traverse a capillary conduit. Whenthe time for traverse exceed the previous time by a certain percentage,coagulation is deemed to have occurred. The apparatus includes anunclosed entry port which is connected to two conduits, the firstreceiving the sample to be assayed, the second receiving overflowsample.

[0018] U.S. Pat. Nos. 5,447,440 and 5,628,961, both incorporated hereinby reference, disclose a single-use cartridge and reader used incoagulation assays. The condition of the sample is determined by itsflow properties as detected, for example, by a conductivity sensor.

[0019] U.S. Pat. Nos. 5,916,522 and 5,919,711 disclose a device whichuses ion-specific electrodes to measure ionic activity of fluidsincluding bodily fluids. The fluids are metered and transported withinthe device by centrifugation and pressurization of the device.

[0020] There remains a need for the apparatus and method of conductingassays of the present invention. This invention is responsive to changesin the coagulation of a blood sample, it can be used at the point ofcare, especially locations, such as a doctor's office, which have noimmediate access to a centralized testing facility, and the apparatuscan be produced in part by microfabrication methods and is readilyadapted to include a multiplicity of tests, including blood gas andanalyte testing.

SUMMARY OF THE INVENTION

[0021] It has now been surprisingly discovered that the needs enumeratedabove, and more, can be fulfilled by the apparatus and method of thepresent invention. In a preferred embodiment of the invention, adisposable, single-use cartridge is disclosed which, along with anexternal reading device, is capable of providing information relating tothe propensity of a fluid sample to undergo changes in viscosity. Inparticular, diagnostic data on biological fluids, can be obtained suchas clotting characteristics of whole blood samples.

[0022] Most importantly, the apparatus and method disclosed can includea battery of tests, all of which can be conducted simultaneously on asingle fluid sample, usually in a matter of tens of seconds. Forexample, the time required to perform a normal PT test is about 12seconds, while about 300 to over 1000 seconds may be needed for ACTtests using the blood from highly heparinized patients. The apparatusand method of the present invention is preferably adapted to make use ofmicrofabrication methods and devices, especially microfabricatedelectrochemical sensors, to allow optimum cartridge configuration andreproducible data acquisition, handling, processing, and storage.

[0023] Coagulation in blood or plasma occurs when fibrinogen isenzymatically converted to fibrin. In this conversion, small peptidefragments are cut from the fibrinogen molecule to produce individualfibrin strands. The strands then form a hydrogen bonded network thatserves to gel the sample. The enzyme responsible for liberation of thefibrinopeptides is the protease thrombin. It is generated in its activeform as the penultimate step in the “coagulation cascade”, a series ofsequential protease activations involving nine plasma proteins.

[0024] Thrombin is a protease that hydrolyses peptides at the carboxylterminal of arginine. Its presence, therefore, can be determined byaddition of an arginine containing substrate which, upon conversion,generates a colored, fluorescent, or electroactive species. In thebroadest aspect of the invention a sensor detects the changes, for anexample, an electrode in the cartridge is used to amperometricallydetermine the liberated electroactive species. Appearance of theelectroactive species is closely correlated with coagulation of thefluid sample.

[0025] Thus, in its most general sense, one embodiment of the presentinvention relates to a cartridge for measuring a change in thecoagulation parameters of a fluid sample comprising: (a) a housingcapable of being charged with a fluid sample and equipped with a sampledisplacement means for applying a force against the fluid sampleeffective to displace at least a metered portion of the fluid samplewithin the housing; (b) at least one substrate, contained within thehousing, capable after contact with the fluid sample of promotingenzymatic reactions related to the coagulation of the fluid sample; (c)at least one sensing means, contained within the housing, capable ofdetecting the enzymatic reactions in the fluid sample. In thisapplication, the term “coagulation parameters” refers to the measurementdetermined by the APTT, PT, ACT and other tests generally related toclot formation, generally quantified as a time to clot formation.

[0026] In particular embodiments of the present invention the housing isequipped with one or more connecting means for engaging the housing witha reading device. For example, the cartridge may have electromechanicalconnectors to allow the cartridge to be engaged to an external readingdevice that performs a variety of functions including, but not limitedto, recording, displaying, manipulating, storing or, otherwise,utilizing the measurements that can be carried out using the cartridgeof the invention.

[0027] In the present invention, the cartridge is equipped with a pumpfor displacement of the fluid sample. For instance, the cartridge may beconnected to an external pump capable of then exerting a force againstthe fluid sample to move the sample within the housing. Alternatively,the sample displacement means may be a pump that already forms anintegral part of the cartridge. In any event, actuation of the sampledisplacement pump allows at least a portion of the fluid sample to moveacross the sensor.

[0028] In a preferred embodiment of the invention the force that isapplied to the fluid sample, as well as its subsequent movement, isreversible so that at least a portion of the fluid sample is displacedback and forth across the sensing means in a substantially reciprocatingmanner. On contact of the fluid sample with the reagent, the subsequentchanges in the thrombin content of the fluid sample are then detected bymonitoring the fluid sample.

[0029] In a specific embodiment of the present invention, an apparatusis disclosed for conducting an assay that is responsive to coagulationof a fluid sample comprising: (a) at least one sensor sensitive to thedisplacement of a fluid sample across the sensor; (b) at least onesensor capable of detecting amperometrically an electroactive species,(c) at least one reagent capable of promoting coagulation of a fluidsample; (d) a substrate capable of reacting with an enzyme associatedwith coagulation with the generation of an electroactive species and (e)a pump for applying pressure against a fluid sample in the sampleretainer to displace at least a portion of the fluid sample across thesensors. Preferably, the force or pressure is applied reversibly tocause the fluid sample to move in a substantially reciprocating manner,such that the fluid sample dissolves the substrate and reagent thatpromotes the coagulation. In particular embodiments of the presentinvention, a pump is provided which comprises a resilient diaphragm influid communication with the sample which provides a pneumatic force tothe fluid sample. A preferred diaphragm pump may have an internal springor an internal rubber sponge to promote the rapid, reproduciblecompression and decompression of the diaphragm.

[0030] The cartridge of this invention has provisions for receiving ablood, plasma, or other fluid sample and for precisely metering apreselected sized aliquot of the fluid sample for further processing.Such a metered aliquot is placed in contact with a premeasured amount ofreagent for activating and for detecting the reactions associated withcoagulation.

[0031] As mentioned previously, the present invention also providescartridges and methods of their use in which the cartridges may becoupled to an external reading device that performs-a number offunctions. Hence, the present invention also relates to an apparatus inwhich the sensor provides a signal to an external reading device thatactuates a plunger for compressing and decompressing the diaphragm pump.Where the sensor is a conductivity (conductimetric) sensor, preferably amicrofabricated conductivity sensor, the signal is a conductivityoutput. In one embodiment, output signals below a first preselectedvalue cause the reading device to actuate the plunger to compress thediaphragm, and output signals above a second preselected value cause thereading device to actuate the plunger to decompress the diaphragm. Inaddition to providing a feedback methodology, the external readingdevice may also provide signal processing capability in which raw datamay be processed to enhance the amount of useful information that may beobtained from a given assay. The external reading device also operatesan amperometric sensor which oxidizes or reduces the electrogenicspecies reaction product which is indicative of coagulation. Thiselectrochemical reaction generates a current which is recorded andprocessed by the external reading device. Another aspect of the presentinvention is the maintenance of the cartridge at a given temperature,preferably at physiological temperature in order to insure a reliableand reproducible coagulation assay.

[0032] Various fluid samples may be assayed according to the presentinvention, including, but not limited to, biological fluids, such aswhole blood and plasma. The present invention is also particularlyuseful for conducting assays on anticoagulated blood samples, including,but not limited to, heparinized or citrated whole blood.

[0033] It is, therefore, an object of the present invention to providean apparatus for conducting a blood test for prothrombin time (PT)comprising: (a) at least one conductivity sensor sensitive to thedisplacement of a blood sample across the sensor; (b) a second sensorcapable of detecting amperometrically an electroactive species; (c) atleast one reagent mixture comprising thromboplastin and calcium ions;(d) a substrate capable of reacting with thrombin with the generation ofan electroactive species; (e) a pump for reversibly applying pressureagainst the blood sample to displace at least a metered portion of theblood sample into contact with the reagent and substrate andsubsequently across the sensors, preferably, in a substantiallyreciprocating manner, the reagent contacting and promoting thecoagulation of the blood sample.

[0034] It is another object of the present invention to provide anapparatus for conducting a blood test for activated partialthromboplastin time (APTT) comprising: (a) at least one conductivitysensor sensitive to the displacement of a blood sample across thesensor; (b) a second sensor capable of detecting amperometrically anelectroactive species; (c) at least one reagent mixture comprising aphospholipid and calcium ions; (d) a substrate capable of reacting withthrombin with the generation of an electroactive species; and (e) a pumpfor reversibly applying pressure against the blood sample to displace atleast a metered portion of the blood sample into contact with thereagent and substrate and subsequently across the sensors, preferably,in a substantially reciprocating manner, the reagent contacting andpromoting the coagulation of the blood sample.

[0035] It is another object of the present invention is to provide anapparatus for conducting a blood test for activated clotting time (ACT)comprises; (a) at least one conductivity sensor sensitive to thedisplacement of a blood sample across the sensor; (b) at least onesensor capable of detecting amperometrically an electroactive species;(c) at least one reagent capable of activating the extrinsic coagulationcascade; (d) a substrate-capable of reacting with an enzyme associatedwith coagulation with the generation of an electroactive species; and(e) a pump for reversibly applying pressure against the blood sample todisplace at least a metered portion of the blood sample into contactwith the reagent and substrate and subsequently across the sensors,preferably, in a substantially reciprocating manner, the reagentcontacting and promoting the coagulation of the blood sample.

[0036] A further object of the present invention is the disclosure of amethod of conducting a coagulation assay comprising: (a) placing a fluidsample in a sample retainer for retaining the fluid sample out ofcontact with a sensor and a reagent, the sensor sensitive to thedisplacement of the fluid sample across the sensor and the reagentcapable of promoting a change in the viscosity of the fluid sample; (b)applying pressure against the fluid sample in the sample retainer todisplace at least a portion of the fluid sample across the sensor.Preferably, the force or pressure is applied reversibly such that thefluid sample moves in a substantially reciprocating manner, such thatthe fluid sample contacts the reagent that promotes the viscosity changeof the fluid sample; (c) detecting the displacement of the fluid sampleacross the sensor to indicate a change in the viscosity of the fluidsample; and (d) detecting the generation of a electroactive species.

[0037] Another object of this invention is to provide a cartridge fordelivering a metered sample to an analysis location comprising: ahousing having a closable sample entry port for receiving an unmeteredfluid sample; a holding chamber having a first end in communication withthe entry port, the holding chamber having a second end with a capillarystop; an analysis location in communication with the capillary stop, thecapillary stop selectively allowing passage of a sample from the holdingchamber to the analysis location; an overflow chamber in communicationwith the holding chamber for handling overflow of incoming sample; and apump for providing a force to the fluid sample in the holding chamber,thereby allowing passage of the sample through the capillary stop.

[0038] Another object of the present invention is to provide a cartridgefor delivering a metered fluid sample to an analysis location,comprising: a housing containing a fluid path and having first andsecond sides, wherein at least one side contains at least one fluidchannel, said first and second sides attached with a wall locatedtherebetween, said wall and said channels providing the fluid path; anda hydrophobic layer comprising a portion of the fluid path, thehydrophobic layer preventing flow of a fluid toward an entry port.

[0039] Another object of the present invention is to provide a cartridgeadapted for use with an analyzer for assaying an enzyme in a fluidsample comprising: a housing having a sample entry port, overflowchamber, holding chamber, and analysis location, an airtight entry portclosure, a pump actuated by the analyzer for moving the sample withinthe cartridge, one or more reagent deposits in the analysis locationcomprising at least one substrate capable of reaction with an enzyme inthe fluid sample, the reaction of the enzyme forming a detectablereaction product, a first sensor for detecting the location of the fluidsample, and a second sensor for detecting the detectable reactionproduct.

[0040] Another object of the present invention is to provide a cartridgeadapted for use with an analyzer for assaying an enzyme in a fluidsample comprising: a housing having a sample entry port, holdingchamber, and analysis location, an airtight entry port closure, a pumpactuated by the analyzer for moving the sample within the cartridge, oneor more reagent deposits in the analysis location comprising at leastone substrate capable of reaction with an enzyme-in the fluid sample,the reaction of the enzyme forming a detectable reaction product, ahydrophobic layer comprising a portion of the analysis location, a firstsensor for detecting the location of the fluid sample, and a secondsensor for detecting the detectable reaction product.

[0041] Another object of the present invention is to provide asingle-use cartridge used in combination with an analyzer fordetermining a coagulation parameter of a sample of blood or bloodderivative comprising: a cartridge having an entry port for receiving anunmetered sample, an entry port closure, a holding chamber incommunication at a first end with the entry port, and a capillary stopin communication with the holding chamber at a second end, the capillarystop also in communication with an analysis chamber, the holding chamberin communication with an overflow chamber for receiving and retainingexcess sample, the overflow chamber in communication with a pneumaticpump actuated by the analyzer, the analyzer actuating the pneumatic pumpto displace sample in the holding chamber through the capillary stopinto the analysis chamber to deliver a metered portion of the sampleinto the analysis chamber, the analysis chamber containing a substratefor the enzyme thrombin capable of dissolving in the metered sample, anamperometric sensor for detecting the product of the reaction betweenthrombin and the substrate, and a conductimetric sensor for detectingthe position of the sample in the analysis chamber, the amperometricsensor and the conductivity sensor connected to the analyzer forproviding output signals to the analyzer, the analyzer capable of usingthe output signal of the conductivity sensor to actuate the pneumaticpump to control the position of the sample in the analysis chamber, theanalyzer capable of determining the coagulation parameter from theoutput signal of the amperometric sensor, and the cartridge containing ahydrophobic region between the capillary stop and the analysis chamberto prevent sample in the analysis chamber from being drawn back into theholding chamber.

[0042] Another object of the present invention is to provide a method ofassaying an enzyme in a sample of blood or blood derivative comprisingthe steps: obtaining a sample of blood or blood derivative, placing thesample into the entry port of a cartridge, closing the entry port,activating the pneumatic pump, thereby forcing a metered sample from thesample chamber into the analysis chamber, oscillating the sample backand forth in the analysis chamber, and determining the concentration ofthe reaction product using the second sensor.

[0043] Another object of the present invention is to provide a method ofassaying an enzyme in a sample of blood or blood derivative comprisingthe steps: introducing the sample into a cartridge, metering a portionof the sample, moving the metered sample to an analysis location, mixingthe metered sample with reagent at the analysis location, allowing theenzyme to react with the reagent, positioning the reacted sample at asensor, and detecting the product of the enzyme reaction using a sensor.

[0044] The present invention further encompasses a disposable,single-use cartridge comprised of a plurality of microfabricated sensorsfor the determination of the presence or concentration of one or moreanalytes in a sample fluid, along with a microfabricated sensor for thedetermination of changes in the viscosity of the sample fluid as well asa microfabricated sensor for the determination of the presence of anelectroactive species.

[0045] Other objects of the present invention will be evident to thoseof ordinary skill, particularly upon consideration of the followingdetailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0046]FIG. 1 is a diagrammatic representation of the coagulationcascade.

[0047]FIG. 2 is a plan view of the upper side of the cartridge.

[0048]FIG. 3 is a cross-sectional view of the sample entry port area ofthe cartridge.

[0049]FIG. 4 is a cross-sectional view of holding chamber and pre-sensorchamber area of the cartridge.

[0050]FIG. 5 is a perspective view of the overflow chamber of thecartridge.

[0051]FIGS. 6A, 6B, and 6C show the oscillation of sample in theanalysis location.

[0052]FIG. 7 shows the conductimetric and amperometric sensors of thecartridge.

[0053]FIG. 8 shows the hydrophobic chip in the fluid path.

[0054]FIG. 9 is a diagram showing the concentration of reagent in thesample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The following describes the invention of a disposable cartridgefor use at the patient bedside to perform traditional coagulation. Thecartridge provides a means by which a blood sample can be metered andquantitatively mixed with reagents that activate the coagulationcascade. Clot formation is subsequently detected using a microfabricatedsensor housed within the cartridge. The functional features of thecartridge and sensor are described.

[0056] U.S. Pat. No. 5,096,669, incorporated by reference, discloses asystem for near-patient testing comprised of a hand held analyzer anddisposable test cartridges. The cartridge is assembled from moldedcomponents and houses the necessary reagents, calibrants, and sensors toperform a variety of clinical chemistry tests. Cartridges containvarious combinations of traditional electrochemical sensors which havebeen miniaturized through microfabrication techniques. Lithographicprocesses and inventive dispensing technology are utilized to create ionselective (Na⁺, K⁺, Cl⁻, NH₃ ⁺, Ca²⁺,pH, and CO₂), amperometric(glucose, creatinine, lactate, oxygen), and conductimetric (hematocrit)sensors. The sensors are packaged so that single cartridges accommodatethe most common testing patterns.

BACKGROUND

[0057] Coagulation testing comprises a number of tests that are used toindicate the health of a patient s coagulation system. Testing is usedto monitor patients receiving acute or prophylactic anti-coagulationtherapy or to screen the patient for coagulation abnormalities. Becausethe process of coagulation is complex and involves a number of bloodcomponents, several coagulation tests have been developed to probe theintegrity of the various coagulation subsystems.

[0058] Blood clotting is the trauma-induced formation of an insolublegelatinous plug that serves to decrease blood flow in the effected area.The gel is formed as fibrin strands, which are generated by the actionof thrombin on the plasma protein fibrinogen, cross-link to form a threedimensional structure. Conversion of fibrinogen occurs as the final stepin a series of enzyme reactions in which sequential coagulation enzymes,or factors, are activated. The series of enzyme reactions is called thecoagulation cascade and is depicted in FIG. 9. It is initiated throughthe activation of Factors XII or VIII. In vivo, the former is convertedto its active form at the surface of platelets that have agglomerated atsites of tissue damage. Factor VIII is activated by thromboplastin, asubstance released by damaged endothelial cells. Activation of FactorXII and Factor VIII is referred to as intrinsic and extrinsicactivation, respectively.

[0059]FIG. 1 is a diagrammatic representation of the enzymatic stepsoccurring in the coagulation cascade. Each factor is converted to itsactive form only during active coagulation. The arrows indicate thesequence of activations required for clot formation. As indicated, manyof the reactions also require the presence of free calcium ions (Ca⁺⁺)and phospholipids (PL), jointly depicted as 82 in FIG. 1. Coagulationcan be initiated through the activation of the intrinsic pathway 74which involves the activation of Factor XII. Another way of initiatingcoagulation is through the extrinsic pathway 60 which causes activationof Factor XII. Activation of Factor XII is depected at 62; of Factor XIat 64; of Factor IX at 66; of Factor X at 68 and at 70; of Factor XIIIat 72; of Factor II at 76 (also termed the conversion of prothrombin tothrombin); and the conversion of soluble fibrinogen to insoluble fibrinat 78 with the formation of a clot at 80.

[0060] The most frequently performed coagulation tests measure the timerequired for clot formation in a blood or plasma sample subsequent tothe addition of activating reagents. The initiation reagent useddictates the portion of the coagulation cascade that will be activatedand therefore assessed. Negatively charged surfaces mimic activation byplatelets and pure tissue thromboplastin is added to initiate clottingvia the intrinsic pathway. The most sophisticated laboratory instrumentsautomatically measure, dispense, and mix reagents and sample. Lessautomated methods require that the user measure and mix the sample andreagent. Clot formation in these instruments is detected eithermechanically or optically.

[0061] The operational specification describes the sequence of eventsthat must occur in the course of a test cycle. For assaying an enzyme ina sample of blood or blood derivative this specification discloses thefollowing method:

[0062] introducing the sample into a cartridge,

[0063] metering of a portion of the sample,

[0064] moving the metered sample to an analysis location,

[0065] mixing the metered sample with reagent at the analysis location,

[0066] positioning the reacted sample at a sensor, and

[0067] detecting the product of the enzyme reaction using a sensor.

[0068] The performance specification sets the criteria for parameterssuch as the range of results that will be reported, the necessaryaccuracy and precision of the test, and the acceptable operatingconditions. The test results must match the sensitivity and range of thecommonly accepted coagulation tests and must do so with comparable orbetter precision. Furthermore, as a point-of-care device may be operatedby non-technically trained personnel, the analyzer software must detectany cartridge errors that do occur.

[0069]FIG. 2 is a plan view of the top or first side 40 of the cartridgeor housing 10. The sample entry port 12 is shown and it is surrounded bya circumferential excess sample well 14. A snap cover 38 closes thesample entry port 12 with the formation of an air-tight seal. A sampleholding chamber 20 is in communication with the sample entry port 12. Acapillary stop 22 is at the end of the sample holding chamber which isdistal to the sample entry port 12. A pre-sensor channel 24 leads fromthe capillary stop 22 to the analysis location 31. A deposit of reagentand substrate 30 is located in the analysis location 31. In addition, incommunication with the analysis location 31, there are conductimetricsensors 28, a amperometric sensor 29, and a reference sensor 32. Theamperometric sensor 29 is located distal to the capillary stop 22 andthe conductimetric sensors 28 are located proximal to the capillary stop22. The analysis location 31 is in communication with the waste tube 34.A hydrophobic layer 26 is located between the pre-sensor channel 24 andthe analysis location 31. The fluid path 38 consists of the sample entryport 12, the holding chamber 20, the capillary stop 22, the pre-sensorchannel 24, the analysis location 31, and the waste tube 34. A flexiblediaphragm pump 36 pumps air which is transmitted through the air tube 18into the overflow chamber 16.

[0070] Although the pump in FIG. 2 is a flexible diaphragm pump, anysuitable pump may be used, such as piston and cylinder, electrodynamic,and sonic.

[0071] Sample. The coagulation assays commonly performed with thecartridge of this invention use a sample of blood, or a sample of ablood derivative such as blood containing an additive or diluent,plasma, serum, or plasma or serum containing an additive or diluent.

[0072] Sample Introduction. The sample is deposited in the cartridgethrough the sample entry port 12 in FIG. 2 and shown in cross-section as12 in FIG. 3. The opening is designed so that capillary forces will drawa hanging drop touched to the orifice of the port into the cartridge andtoward the sample holding chamber. The action is a result of thegeometry and high surface energy of the plastic conduit. A high surfaceenergy is achieved with a corona treatment or equivalent treatment, suchas an ion-plasma treatment, before cartridge assembly. Once bloodreaches the sample holding chamber, its geometry and corona-treatedsurface causes the blood to pass along its length up to the point of acapillary stop. The upper limit of the cross-sectional area of thesample holding chamber is that which would prevent capillary draw if acartridge were to be held upright as it was filled. The lower limit ofthe cross-section is determined by the sample volume required fortesting and the reproducibility required of this volume. The sampleholding chamber contains 19 microliters with a cross-sectional area of0.0075 cm². In other embodiments the volume of the metered fluid sampleis in the range of 1 microliter to 1 milliliter. A preferred volume ofthe metered fluid sample is in the range of 15 microliters to 50microliters.

[0073] Metering the fluid sample. The reproducibility of the volume ofsample that is moved into the sensor channel for mixing affects thereproducibility of the final concentration of dissolved reagent in theblood. The metering method described in the following providesvolumetric reproducibility.

[0074] In all cartridges the metering and pumping of fluids and reagentsis independent of the user. Once the user fills the cartridge, shuts thesnap closure to seal the blood entry port and inserts the cartridge intothe analyzer, the test cycle occurs and is monitored through theanalyzer software. Upon insertion, the analyzer first recognizes thecartridge type. The appropriate test sequence is initiated and thetiming, speed, and duration of all subsequent fluid motions arecontrolled. The blood sample is moved into the sensor channel, alsotermed the analysis location 31 in FIG. 2 once the heating elementsstabilize at 37° C. The blood sample is moved forward as a piston in theanalyzer pushes on the membrane 36 of the diaphragm pump. This forcesair through the air pipe 18 that connects the air bladder and theoverflow chamber 16 and moves the blood that is in front of the orificetoward the sensor channel. The volume of the metered fluid sample is thevolume of the holding chamber 20 between the orifice (48 in FIG. 5) inthe wall of the holding chamber and the capillary stop 22. The volume ofblood that is moved will depend primarily on the volume of blood infront of the orifice and secondarily on the surface area-to-volume ratioof the sample-holding chamber, the sample hematocrit (the percent of theblood volume comprised of red blood cells), and the fluid speed. Theselatter three parameters determine the volume of sample that will remainon the walls of the sample holding chamber as the chamber is evacuated.The fluid will be metered most precisely at low velocity from a chamberwith a low surface-area-to-volume ratio. The lower limit on the sampleholding chamber cross-sectional area is determined by the allowablevariation in the volume loss to shear at the necessary fluid speed.

[0075]FIG. 3 is a cross-sectional view of the sample entry port 12 areaof the cartridge or housing 10. FIG. 3 shows the top or first side orupper housing 40 of the cartridge, the base or second side or lowerhousing 44 of the cartridge, and the wall or tape or film 42 interposedbetween the first and second sides. The tape 42 has an adhesive layer oneach side and adheres to the top 40 and base 44 sides of the cartridge.The sample entry port 12 is shown filled with sample 46, and the sample46 has also filled the sample holding chamber 20. A circumferential well14 surrounds the sample entry port 12 and is show filled with excesssample.

[0076] To precisely fill the sample holding chamber, there must besufficient capillary draw to avoid under filling the cartridge as wellas a stop feature that prevents sample from overflowing into thepre-sensor channel. As depicted in FIG. 4, a capillary stop 22 is formedby a small bore or through-hole in the tape gasket 42 betweenoverlapping sections of the sample holding chamber 20 and the pre-sensorchannel 24 serves this purpose. The capillary stop 22 that is formed isrelatively short; only the thickness of the tape 42 gasket. Althoughthis decreases the resistance of the capillary and thereby decreases itseffectiveness in stopping the fluid once the cartridge fills, it isnecessary as it minimizes the high shear zone through which the samplemust pass as it is pushed through the bore for delivery into thepre-sensor channel. The low volume high-shear region minimizes the lossof sample to the walls of the capillary and decreases the potential forthe inclusion of entrapped air segments as the back end of the movingfluid column exits the capillary region.

[0077]FIG. 4 is a cross-sectional view of the conjunction of the sampleholding chamber 20 and the pre-sensor chamber 24 and the capillary stop22. The base 44 is shown with the sample holding chamber 20 cut into it.The pre-sensor channel 24 is shown cut into the top 40. The tape 42forms the top wall of the sample holding chamber 20 and the bottom wallof the pre-sensor chamber 24. A capillary bore or through-hole 22pierces the tape 42 and restricts flow between the sample holdingchamber 20 and the pre-sensor chamber 24.

[0078] The capillary stop of FIG. 4 is a circular bore or through-hole.Other suitable shapes for the capillary stop include rectangular andirregular in shape. If rectangular in shape, suitable examples have asmallest dimension of about 100 microns to about 400 microns. In suchexamples, the largest dimension of the capillary stop is about 100microns to about 1000 microns.

[0079] The capillary stop is of sufficient resistance to stop capillarydraw into the pre-sensor channel. It is not sufficient to resist suddenpressure changes that occur as the cartridge closure is snapped shut. Toreduce the force at the capillary opening at this point, two “overflow”features are incorporated within the cartridge. The first is theoverflow well 14 in FIGS. 2 and 3. As the snap closure is shut, someexcess sample is pushed into the well rather than further into thecartridge. The second feature is an orifice 48 (in FIG. 5) or pressurevent through which excess sample may flow into the overflow chamber 16.As depicted in FIG. 5, the overflow chamber 16 is a low volume chamberin the cartridge top side located above the sample holding chamber andseparated from the chamber by a tape 42 wall. An orifice 48 in the tape42 allows flow of excess sample into the overflow chamber. The hole ororifice 48 in the tape gasket 42 has a greater area than the opening ofthe capillary stop and therefore the orifice has lower flow resistancethan does the capillary stop. The overflow chamber 16 above the tapeopening or orifice 48 has very low walls so that once sample is pushedthrough this hole, it touches the corona-treated plastic and is drawninto the chamber. The sample displaced as the cartridge is closed istherefore trapped within this chamber. When the air bladder iscompressed, air is forced through the air pipe 18 into the overflowchamber 16. The high surface area to volume ratio of this regionencourages sample shear so that the air pushes a path through the excesssample leaving the excess sample on the walls of the overflow chamber.

[0080]FIG. 5 is a perspective view of the overflow chamber. 16. Theoverflow chamber 16 is located directly above the sample holding chamber(not shown in FIG. 4). Tape 42 which is the top wall of the sampleholding chamber (20 in FIG. 2) also forms the bottom wall of theoverflow chamber 16. An orifice 48 in the tape 42 allows communicationbetween the overflow chamber 16 and the sample holding chamber (20 inFIG. 2). The orifice may be circular, rectangular, or irregular inshape. The overflow chamber is constructed in the form of a low box. Anair tube 18 delivers air from the pump 36 (not shown in FIG. 5) to theoverflow chamber 16. The volume of the overflow chamber is in the rangeof 0.2 microliters to 1 milliliter. A preferred volume of the overflowchamber is in the range of 1 microliter to 10 microliters. The diameterof the circular orifice is from about 100 microns to about 1000 microns.

[0081] Movement of sample. In FIG. 2, a metered sample is forced fromthe sample holding chamber 20 through the pre-sensor channel 24 into theanalysis location 31 as the plunger in the analyzer compresses thecartridge air bladder 36 and forces air through the air pipe 18 into theoverflow chamber 16 and through the orifice (48 in FIG. 5). As thesample moves through the dry conduits, it is necessary that the fluidfront wet the walls of the channels uniformly. If the surface energy ofthe conduit is not equal on all sides, uneven flow may occur, causingthe formation of air bubbles within the segment. The surfaces of thechannel within the cover, the adhesive gasket, the reagent coating, andthe chips must therefore be of equivalent surface energy. Surfacetreatments of the components are needed to assure this uniformity.

[0082] Reagent. It is well known in the art to place dried reagent in afluid path for reaction with a sample to be assayed. A variety ofcomponents are included in the reagent, some of which contribute to therapid redissolving of the dried reagent by the fluid sample. Theseinclude a water-soluble polymer, gelatin, agarose, a polysaccharide,polyethylene glycol, polyglycine, a saccharide, sucrose, an amino acid,glycine, a buffer salt, sodium phosphate, HEPES buffer, and a dyemolecule.

[0083] It is known in the art to include a material for inducingcoagulation via the extrensic pathway (60 in FIG. 9). Material suitablefor this use with the cartridge of this invention include celite,kaolin, diatomaceous earth, clay, silicon dioxide, ellagic acid, naturalthromboplastin, recombinant thromboplastin, phospholipid, and mixturesthereof. A preferred inducer is celite.

[0084] Thrombin-substrate Reaction. The substrate used in theelectrogenic assay has an amide linkage that mimics the thrombin-cleavedamide linkage in fibrinogen. Specifically, the substrate is atosyl-glycyl-prolinyl-arginyl-, H-D-phenylalanyl-pipecolyl-, orbenzyl-phenylalanyl-valyl-arginyl-moiety attached to anN-phenyl-p-phenylenediamine or N-[p-methoxyphenyl-]-p-phenylenediaminemoiety. Thrombin cleaves the amide bond at the carboxy-terminus of thearginine residue or pipecolyl residue because the bond structurallyresembles the thrombin-cleaved amide linkage in fibrinogen. The productof the thrombin-substrate reaction is the electrochemically inerttosyl-glycyl-prolinyl-arginyl-, H-D-phenylalanyl-pipecolyl-, orbenzyl-phenylalanyl-valyl-arginyl- and the electroactive compoundsN-phenyl-p-phenylenediamine or N-[p-methoxyphenyl-]-p-phenylenediamine.The tripeptide sequence was chosen because it renders the substratevirtually non-reactive with blood proteases other than thrombin and thereactivity of thrombin with the arginine amide linkage in the moleculeis very similar to its reactivity with the target amide linkage infibrinogen. When the substrate is present in a blood or blood derivativesample, generated thrombin simultaneously converts it and fibrinogen totheir cleavage products. The electrochemical species reaction product isdetected by an electrochemical sensor.

[0085] There are a wide variety of suitable electrogenic materials whichexhibit reversible or quasi-reversible electrochemical reactions knownin the art which may be assayed using an amperometric sensor of thisinvention. For example, ferrocene, ferrocyanide, and otherorganometallic species may be detected. Others include phenazinederivatives. Any suitable electrogenic material may be combined with asuitable substrate for use in assaying an enzyme. For example, suitableelectrogenic materials may be combined with a suitable tripeptide withan arginine residue for use in determining the presence of thrombin.

[0086] An indicator electrogenic material which is detected at apotential different from the detection potential for the substrate orthe electrogenic product of the enzymatic reaction may be included inthe reagent. Such a second electrogenic material is useful forstandardizing the amperometric sensor. Suitable electrogenic materialsfor this purpose include ferrocene, ferrocyanide, and otherorganometallic species, phenazine derivatives,N-phenyl-p-phenylenediamine and N-[p-methoxyphenyl-]-p-phenylenediamine.

[0087] The test is termed “electrogenic” because the electrochemicallydetectable species is generated to allow determination of a ratemeasurement or the test endpoint. This is similar to “chromogenic” or“fluorogenic” endpoint tests in which a change in the light absorbing oremitting properties of a sample indicates the rate measurement orendpoint. In a chromogenic test, for example, the cleaved portion of thesubstrate molecule is colorless when attached to the tripeptide andbrightly colored when liberated by the action of thrombin. By monitoringthe wavelength at which the free species absorbs light, the time atwhich active thrombin is produced can be determined. chromogenic APTTand PT tests have been shown to have good correlation to traditionalAPTT and PT plasma tests.

[0088] The cartridge of this invention is not limited only to the assayof coagulation enzymes. Assays can be devised for a variety of enzymes,such as glucose oxidase, lactate oxidase, and other oxidoreductases,dehydrogenase based enzymes, and alkaline phosphatase and otherphosphatases, and serine proteases. Other enzymes known in the art to beassayed in clinical chemical procedures can be assayed with thisinvention.

[0089] Reagent Mixing. Once in the analysis location, the sample must bemixed with the reagent. For these tests, it is required that the reagentis homogeneously distributed throughout the sample in the region of thesensor within a few seconds of the start of dissolution.

[0090] In the coagulation cartridge, the clot reaction is initiated in aspecific region of the sensor channel over the sensor chips. A length ofthe wall within the channel is coated with the reagent, as indicated at30 FIG. 2 and FIG. 6A-C. Oscillating a segment of the sample over thereagent induces convection. The motion is controlled so that thetrailing edge of the blood segment continually moves back and forthacross the reagent coating, as depicted in FIG. 6A-C.

[0091] FIGS. 6A-C show the analysis location 31 along with otherportions of the fluid path pre-sensor channel 24 and waste tube 34. Thedried reagent deposit 30 is shown in the analysis location 31. FIG. 6Bshows a sample 46 which has been moved past the reagent deposit. FIG. 6Cshows the sample 46 after it has been oscillated back over the areawhere the reagent was deposited. Although the reagent 30 is showndeposited in the analysis location 31 in FIG. 6A, it is possible toplace the reagent deposit at more than one site in the analysislocation, and reagent deposits may be placed at any location in theentire fluid path (38 in FIG. 2).

[0092] The oscillation is maintained using a fluid position sensorcoincident with the reagent coating. This sensor comprises the twoparallel bars on the sensor chip shown in FIG. 6. FIG. 6 shows theconductimetric sensors 28 . These sensors 28 lie perpendicular to thelength of the sensor channel and the electrical resistance between themis used to monitor the relative position of the fluid front. At theextremes, an open circuit reading indicates that the fluid has beenpushed off the sensor and a closed circuit reading indicates the sensoris covered with sample. The fluid is continually moved forward and backat a controlled velocity. Controlling the time for which the sensorremains open and closed circuit controls the position at which the fluidchanges direction.

[0093] In a preferred method, the pneumatic pump oscillates the samplein the analysis chamber with the trailing edge of the sample positionedin the region of the conductivity sensor in order to dissolve thesubstrate in that portion of the sample near the trailing edge. Theoscillation may be at a frequency in the range of 0.2 to 10 Hertz for aperiod in the range of 1 to 100 seconds. In a preferred method, theoscillation is at a frequency in the range of about 1.5 Hertz for aperiod of about 20 seconds. In another preferred method the oscillationis at a frequency of about 0.3 Hertz and the amperometric or secondsensor generates a signal at each oscillation. If erythrocytes arepresent in the fluid sample, the oscillation is at a frequency adequateto prevent the settling of erythrocytes on the sensor. In a preferredmethod, the amperometric sensor determines the concentration of theproduct each time the sample is oscillated past the amperometric sensor.

[0094] In a preferred embodiment, the first amperometric sensor signalis stored by the analyzer and subsequent signals from the amperometricsensor are stored and are compared to the first and other stored signalsin order to determine the maximum rate of change in the amperometricsensor signal. These data are analyzed to determine a fixed fraction ofthe maximum rate of change of the amperometric sensor signal. These dataare used to determine the coagulation parameter of interest.

[0095]FIG. 7 also shows the amperometric sensor 29 in which the sensingportion is in the form of an antenna 31.

[0096] Although the sensor in the example in FIG. 7 is an amperometricsensor, other electrochemical processes which use other electrochemicalsensors can be used. For example, a potentiometric sensor may be used todetect ion species such as N^(a+) and K⁺.

[0097] In the preferred embodiment of the present invention the analyzerapplies a potential to a amperometric sensor at with the generation ofan electrochemical signal, said signal being proportional to theconcentration of the product in the fluid sample. The amperometricsensor has an applied potential of approximately +0.4 V versus asilver-silver chloride electrode and, in another preferred embodiment,the amperometric sensor has an applied potential of approximately +0.1 Vversus a silver-silver chloride electrode. The signal generated by theenzyme reaction product at approximately +0.1 V is distinguishable fromthe signal generated by the unreacted substrate at approximately +0.4 V.

[0098] In the embodiments of the invention which use the substratestosyl-glycyl-prolinyl-arginyl-, H-D-phenylalanyl-pipecolyl-, orbenzyl-phenylalanyl-valyl-arginyl-moiety attached to anN-phenyl-p-phenylenediamine or N-[p-methoxyphenyl-]-p-phenylenediaminemoiety, the intact substrates are detected at a voltage of approximately+0.4 V. The electrogenic reaction products N-phenyl-p-phenylenediamineor N-[p-methoxyphenyl-]-p-phenylenediamine are detected at a voltage ofapproximately +0.1 V. Thus in these embodiments, the analyzer applies apotential to a amperometric sensor with the generation of anelectrochemical signal which is proportional to the concentration of thesubstrate in the fluid sample. Also, the analyzer applies a potential toa amperometric sensor with the generation of an electrochemical signalwhich is proportional to the concentration of the product in the fluidsample. After hydrolysis of the substrate by thrombin, a product isformed which reacts at the amperometric sensor with the generation of asignal distinguishable from the signal generated by the substrate.

[0099] It should be noted that the exact voltages used toamperometrically detect the substrate and the product will varydepending on the chemical structure of the substrate and product. It isimportant that the difference in the voltages used to detect thesubstrate and the product be great enough to prevent interferencebetween the readings. With some substrates, the voltage required toelectrochemically detect the substrate is so high as to beyond practicalmeasurement. In these cases, it is only necessary that the product bedetectable amperometrically.

[0100] The sensors are preferably microfabricated of any suitableelectroconductive material and are preferably made of gold, platinum,silver or iridium. The methods for patterning metals on silicon wafersare well known in the art. It is also desirable to coat the sensor witha thin organic layer which prevents poisoning of the sensor surface byblood components such as a self-assembled thiol film, as is known in theart. Mercaptoalkanols form self-assembled thiol firms, and some examplesinclude mercaptoethanol, mercaptopropanol, mercaptobutanol, and mixturesthereof.

[0101]FIG. 8 is a plot of the distance from the end of the samplesegment in mm along the abscissa versus the concentration of dissolvedreagent in micro moles along the ordinate. A diagram at the top of FIG.8 shows a fluid sample 46, conductimetric sensors 28, and amperometricsensor 30. The data points of FIG. 8 indicate the concentration ofdissolved reagent along the length of the column of fluid sample.

[0102] Mixing in this manner produces a concentration gradient along thelength of the blood segment. As shown in FIG. 8, the concentration ishighest at the edge of the segment that was swept across the reagent anddecreases toward the center of the fluid column. In FIG. 8 the measuredconcentration and the cartridge-to-cartridge variability in theconcentration (the error bars show one standard deviation) are plottedas a function of position along the blood segment,. At the sensorlocation, one standard deviation of the reagent concentration is 10% ofthe mean concentration.

[0103] Maintaining thefluid position. In this embodiment the dissolutionprofile is not uniform, cartridge-to-cartridge reproducibility of thereagent concentration at the sensor depends on both thecartridge-to-cartridge consistency of sample positioning and the abilityto maintain quiescence within the sample throughout the course of thetest. The former is achieved through active position control usingfeedback from the same fluid position sensor employed to monitor themixing oscillation. For short duration tests, the resistance between thebars of the sensor is maintained within a window a set number of ohmsabove the closed circuit reading. The sample-air interface is thereforeheld between the two bars. If the sample is drifting back toward thesample-holding chamber, the resistance will decrease until a pre-setlimit is triggered causing the analyzer to push the sample forward untilthe control resistance is again achieved. If the sample drifts towardsthe cartridge waste tube, the resistance will drift higher causing theanalyzer to pull the sample backwards. Because the resistance is asensitive function of the fluid position, the fluid front can bemaintained within 100 microns of a nominal position. The correctivemotions do not cause convection within the sample as they are of verylow amplitude and speed.

[0104] Control to a set resistance is sufficient in tests requiring lessthan 60 to 100 seconds for completion. Some types of coagulation testsproduce results that are always in this range. Other tests, however,require up to 15 minutes before the endpoint is achieved. With extendedtimes, red blood cells may settle and blood components may dry at theexposed chip surface. Both conditions cause the resistance for a givenfluid position to increase and interfere with the position controller.In the case of settling, the resistance gradually increases causing thecontroller to respond as though the fluid has drifted forward. Thesegment is then pulled backward to maintain the set-point resistance.

[0105] To circumvent these problems in tests requiring longer periods ofposition control, the fluid is periodically moved to the fully closedcircuit position and the closed circuit resistance is measured. Thefluid is then re-positioned at a resistance value offset relative to thenew closed circuit reading. The oscillation continually wets the chip toprevent drying and the offset resistance is set relative to the closedcircuit reading for the settled sample. These motions do not causeexcessive convection within the sample as they are again of lowamplitude and velocity.

[0106] Convection occurs within the sample near the sensor if thepositioned segment is not fluidically isolated. Any flow path connectingthe positioned sample segment with the sample-holding chamber willprovide a route by which the sample segment may be siphoned backwardsinto the holding chamber. This is because the surface energy and surfacearea to volume ratio of the sample-holding chamber necessary to allowcapillary draw also causes the chamber to be preferentially wetted. Ifthis occurs, the motion of fluid siphoning from the tail end of thesample segment causes fluid deeper within the segment to be drawn overthe sensor. The reagent concentration in the sample volume around thesensor therefore decreases as it is mixed with sample containing a lowerconcentration of reagent.

[0107] A fluid path which is of sufficiently low resistance to allowthis transfer forms at the seam between the cartridge cover and the tapeas the sample is pushed through the pre-sensor channel. This is becausethe radius of the pre-sensor channel.edge, which is created during theprocess of injection molding the cover, when pressed against the tapegasket may form a thin capillary that is filled by blood sheared fromthe passing segment. Once this capillary is filled, sample may siphonthrough it back into the sample-holding chamber. To prevent siphoning,the fluid path must be broken. In one embodiment, this is accomplishedin the coagulation cartridge by cutting away a section of the tapegasket beneath the pre-sensor channel to expose the surface of apolytetrafluoroethylene chip that is contained within a well in thebase. The surface of the chip is flush with the surface of the base. Thecutout in the tape undercuts the cover channel so that the capillaryformed at the cover-gasket seam is interrupted in the region of thechip. The hydrophobic surface of the polytetrafluoroethylene chipprevents the formation of an alternate pathway along the surface exposedby the opening. This is shown schematically in FIG. 9.

[0108]FIG. 9 shows a polytetrafluoroethylene chip 26, located in a wellon the base and covered by the tape 42, and a portion of the pre-sensorchannel 24. In this portion of the pre-sensor channel 24, the channel iscut into the top or first side of the cartridge and the tape 42 formsthe bottom wall of the pre-sensor channel 24. FIG. 9 shows a section 50where the tape 42 has been cut away exposing a portion 51 of thepolytetrafluoroethylene chip 26 to the sample contained in thepre-sensor channel 24. Sample which has entered the capillary regiondescribed in the previous paragraph is depicted in FIG. 9 at 52.

[0109] The hydrophobic area may be constructed using a number ofdifferent materials. It may be a hydrophobic matrix coating such as wax,petroleum gel, and non-polar organic film. The hydrophobic area may beformed of polytetrafluoroethylene, plastic coated withpolytetrafluoroethylene, polyimide treated with a fluoride ion-plasma,silicon dioxide coated with an organic compound, an alloy of tungstenand titanium, and silver coated with silver chloride. A preferredhydrophobic area is made of polytetrafluoroethylene.

[0110] Prototype cartridges to perform Activated Clotting Time (ACT),Activated Partial Thromboplastin Time (APTT), and Prothrombin Time (PT)tests havwbeen developed and tested. Clinical trials conducted with eachcartridge type have demonstrated satisfactory performance with respectto test precision and accuracy. All coagulation tests utilize the samecartridge components and are differentiated by the composition of thedry reagent.

[0111] It will be apparent to those skilled in the art that the examplesand embodiments described herein are by way of illustration and not oflimitation, and that other examples may be utilized without departingfrom the spirit and scope of the present invention, as set forth in theappended claims.

What we claim is:
 1. A cartridge for delivering a fluid sample to ananalysis location comprising: a housing having a closable sample entryport for receiving a fluid sample; a holding chamber having a first endin communication with the entry port, the holding chamber having asecond end with a capillary stop; wherein the analysis location is incommunication with the capillary stop; and wherein the capillary stopselectively allows passage of the sample from the holding chamber to theanalysis location; an overflow chamber in communication with the holdingchamber for handling overflow of incoming sample; and a pump forproviding a force to the fluid sample in the holding chamber, therebyallowing passage of the sample through the capillary stop.
 2. Thecartridge of claim 1 wherein the overflow chamber is located above theholding chamber and separated from the holding chamber by a holdingchamber wall.
 3. The cartridge of claim 1 wherein the communicationbetween the overflow chamber and the holding chamber is through anorifice in the holding chamber wall.
 4. The cartridge of claim 3 whereinthe shape of the orifice is circular.
 5. The cartridge of claim 3wherein the orifice is circular with a diameter of about 100 microns toabout 1000 microns.
 6. The cartridge of claim 3 wherein the area of theorifice is larger than the area of the capillary stop.
 7. The cartridgeof claim 3 wherein the orifice has a lower resistance to fluid flow thandoes the capillary stop.
 8. The cartridge of claim 3 wherein a roof ofthe holding chamber comprises a tape and the orifice comprises a hole inthe tape.
 9. The cartridge of claim 3 wherein the volume of the holdingchamber between the orifice and the capillary stop correspondssubstantially to the volume of the fluid sample.
 10. The cartridge ofclaim 9 wherein the overflow chamber receives excess sample from theholding chamber through the orifice.
 11. The cartridge of claim 1further comprising a pre-sensor chamber between the capillary stop andthe analysis location.
 12. The cartridge of claim 11 wherein thecross-sectional area of the holding chamber is larger than thecross-sectional areas of the pre-sensor channel and the analysislocation.
 13. The cartridge of claim 11 further comprising a hydrophobicarea between the analysis location and the pre-sensor chamber.
 14. Thecartridge of claim 13 wherein the hydrophobic area comprises ahydrophobic matrix coating selected from the group consisting of wax,petroleum gel, and non-polar organic film.
 15. The cartridge of claim 13wherein the hydrophobic area comprises a layer of material selected fromthe group consisting of polytetrafluoroethylene, plastic coated withpolytetrafluoroethylene, polyimide treated with a fluoride ion-plasma,silicon dioxide coated with an organic compound, an alloy of tungstenand titanium, and silver coated with silver chloride.
 16. The cartridgeof claim 13 wherein the hydrophobic area comprises a layer ofpolytetrafluoroethylene.
 17. The cartridge of claim 1 wherein thecapillary stop has a rectangular shape. wherein the smallest dimensionis about 100 microns to about 400 microns.
 18. The cartridge of claim 1wherein the overflow chamber has walls which are wetted when excesssample enters the overflow chamber.
 19. The cartridge of claim 1 whereinexcess sample is forced into the overflow chamber by closure of theentry port closure.
 20. The cartridge of claim 1 wherein the wallsurfaces of the holding chamber are corona treated.
 21. The cartridge ofclaim 1 wherein the volume of the sample is in the range of 1 microliterto 1 milliliter.
 22. The cartridge of claim 1 wherein the volume of thesample is in the range of 20 microliters to 50 microliters.
 23. Thecartridge of claim 1 wherein the volume of the overflow chamber is inthe range of 0.2 microliters to 1 milliliter.
 24. The cartridge of claim1 wherein the volume of the overflow chamber is in the range of 1microliter to 10 microliters.
 25. The cartridge of claim 1 wherein thepump is in fluidic connection with the overflow chamber.
 26. Thecartridge of claim 1 wherein the force provided to the sample is apneumatic force.
 27. The cartridge of claim 1 which includes an upperhousing, a lower housing, and a film coated on both sides with adhesive,the film interposed between the upper housing and the lower housing. 28.The cartridge of claim 1 wherein the holding chamber or the overflowchamber or both, or portions thereof, are treated to impart a highenergy surface to the interior chamber surfaces.
 29. The cartridge ofclaim 1 adapted for use with an analyzer.
 30. The cartridge of claim 29wherein the pump is actuated by an actuator element of the analyzer. 31.The cartridge of claim 1 further comprising a predetermined amount ofreagent in the analysis location for mixing with the fluid sample. 32.The cartridge of claim 1 further comprising a circumferential wellaround the entry port for receiving spilled fluid sample.
 33. Thecartridge of claim 1 wherein the interior surfaces of the holdingchamber and or the overflow chamber are corona treated.
 34. Thecartridge of claim 1 wherein the holding chamber has a lower interiorsurface to volume ratio than does the overflow chamber.
 35. Thecartridge of claim 1 wherein the entry port closure makes an air-tightseal when closed.
 36. The cartridge of claim 1 further comprising apredetermined amount of reagent in the holding chamber for mixing withthe sample.
 37. The cartridge of claim 1 further comprising at least onesensor.
 38. The cartridge of claim 1 in which the analysis locationcomprises at least one sensor.
 39. A cartridge for delivering a fluidsample to an analysis location, comprising: a housing containing a fluidpath and having first and second sides, wherein at least one sidecontains at least one fluid channel, said first and second sidesattached with a wall located therebetween, said wall and said channelsproviding the fluid path; and a hydrophobic area comprising a portion ofthe fluid path, the hydrophobic area inhibiting flow of a fluid backtoward an entry port.
 40. A cartridge adapted for use with an analyzerfor assaying an enzyme in a fluid sample comprising: a housing having asample entry port, overflow chamber, holding chamber, and analysislocation, an airtight entry port closure, a pump actuated by theanalyzer for moving the sample within the cartridge, one or more reagentdeposits in the analysis location comprising at least one substratecapable of reaction with an enzyme in the fluid sample, the reaction ofthe enzyme forming a detectable reaction product, a first sensor fordetecting the location of the fluid sample, and a second sensor fordetecting the detectable reaction product.
 41. The cartridge of claim40, further including a hydrophobic area comprising a portion of theanalysis location.
 42. The cartridge of claim 40 wherein the reactionproduct is detected by an optical sensor.
 43. The cartridge of claim 40wherein the reaction product is an electrochemical species detected byan electrochemical sensor.
 44. The cartridge of claim 40 wherein theenzyme is selected from the group consisting of factor VII, factor VIII,factor IX, factor X, factor XI, factor XII, and thrombin.
 45. Thecartridge of claim 40 wherein the enzyme is thrombin.
 46. The cartridgeof claim 40 wherein the reagent includes solubility-enhancingcomponents.
 47. The cartridge of claim 40 wherein the volume of thefluid sample delivered to the analysis location is metered.
 48. Thecartridge of claim 40 wherein the reagent includes an electrochemicalspecies other than the substrate and its reaction product.
 49. Thecartridge of claim 48 wherein the electrochemical species is detectableat a different electrical potential than the substrate and the product.50. The cartridge of claim 40 wherein substrate or reagent is depositedat more than one site within the analysis location.
 51. The cartridge ofclaim 40 wherein the fluid sample is oscillated past the first andsecond sensors while in the analysis location.
 52. The cartridge ofclaim 40 wherein the second sensor measures the concentration ofreaction product each time the fluid sample is oscillated past thesecond sensor.
 53. The cartridge of claim 40 wherein the reagentcomprises a matrix that promotes rapid dissolution into the fluidsample.
 54. The cartridge of claim 40 wherein the reagent comprises oneor more components selected from the group consisting of a water-solublepolymer, gelatin, agarose, a polysaccharide, polyethylene glycol,polyglycine, a saccharide, sucrose, an amino acid, glycine, a buffersalt, sodium phosphate, HEPES buffer, and a dye molecule.
 55. Thecartridge of claim 40 further comprising a reagent that promotes thecoagulation of blood or a blood derivative.
 56. The cartridge of claim55 wherein the reagent is selected from the group consisting of celite,kaolin, diatomaceous earth, clay, silicon dioxide, ellagic acid, naturalthromboplastin, recombinant thromboplastin, phospholipid, and mixturesthereof.
 57. The cartridge of claim 40 wherein the first sensor is aconductimetric sensor and the second sensor is an amperometric sensor.58. The cartridge of claim 57 wherein the analyzer applies a potentialto an amperometric sensor with the generation of an electrochemicalsignal, said signal being proportional to the concentration of thesubstrate in the fluid sample.
 59. The cartridge of claim 57 wherein theanalyzer applies a potential to a amperometric sensor with thegeneration of an electrochemical signal, said signal being proportionalto the concentration of the product in the fluid sample.
 60. Thecartridge of claim 57 wherein hydrolysis of the substrate by thrombinforms a product which reacts at the amperometric sensor with thegeneration of a signal distinguishable from a signal generated by thesubstrate.
 61. The cartridge of claim 57 wherein the amperometric sensoris microfabricated.
 62. The cartridge of claim 57 wherein theconductimetric sensor is microfabricated.
 63. The cartridge of claim 57wherein said amperometric sensor has an applied potential ofapproximately +0.4 V versus a silver-silver chloride electrode.
 64. Thecartridge of claim 57 wherein said amperometric sensor has an appliedpotential of approximately +0.1 V versus a silver-silver chlorideelectrode.
 65. The cartridge of claim 57 wherein said conductimetricsensor is proximal to the capillary stop and said amperometric sensor isdistal from the capillary stop.
 66. The cartridge of claim 40 whereinsaid first or said sensor is comprised of a metal selected from thegroup consisting of gold, platinum, silver, and iridium.
 67. Thecartridge of claim 40 wherein said first or said second sensor is coatedwith a self-assembled thiol film.
 68. The cartridge of claim 40 whereinsaid first or said second sensor is in the shape of an antenna.
 69. Thecartridge of claim 40 wherein the reagent includes a substance thatpromotes coagulation of blood.
 70. The cartridge of claim 40 wherein thefluid sample is blood or a blood derivative.
 71. The cartridge of claim70 wherein the blood derivative is selected from the group consisting ofblood containing an additive or diluent, plasma, serum and plasma orserum containing an additive or diluent.
 72. The cartridge of claim 40wherein the substrate is selected from the group consisting of atosyl-glycyl-prolinyl-arginyl-, H-D-phenylalanyl-pipecolyl-, andbenzyl-phenylalanyl-valyl-arginyl-moiety attached to a moiety selectedfrom the group consisting of an N-phenyl-p-phenylenediamine, and anN-[p-methoxyphenyl-]-p-phenylenediamine moiety.
 73. The cartridge ofclaim 40 wherein the reaction product is selected from the groupconsisting of N-phenyl-p-phenylenediamine andN-[p-methoxyphenyl-]-p-phenylenediamine moiety.
 75. The cartridge ofclaim 40 wherein the enzyme is selected from the group consisting ofglucose oxidase, lactate oxidase, and other oxidoreductases,dehydrogenase based enzymes, alkaline phosphatase and otherphosphatases, and serine proteases.
 76. The cartridge of claim 40wherein a sensor is coated with a mercaptoalkanol reagent selected fromthe group consisting of mercaptoethanol, mercaptopropanol,mercaptobutanol, and mixtures thereof.
 77. The cartridge of claim 40wherein a reagent deposit is in the fluid path.
 78. A method of assayingan enzyme in a sample of blood or blood derivative comprising the stepsof: obtaining a sample of blood or blood derivative, placing the sampleinto the entry port of a cartridge of claim 40, closing the entry port,activating the pump, thereby forcing a sample from the sample chamberinto the analysis chamber, oscillating the sample back and forth in theanalysis chamber, and determining the concentration of the reactionproduct using the second sensor.
 79. The method of claim 78 wherein thepump oscillates the sample in the analysis chamber with the trailingedge of the sample positioned in the region of a selected sensor inorder to dissolve the substrate in that portion of the sample near thetrailing edge.
 80. The method of claim 78 wherein the oscillation is ata frequency in the range of 0.2 to 10 Hertz for a period in the range of1 to 100 seconds.
 81. The method of claims 78 wherein the oscillation isat a frequency in the range of about 1.5 Hertz for a period of about 20seconds.
 82. The method of claim 78 wherein the oscillation is at afrequency of about 0.3 Hertz and the second sensor generates a signal ateach oscillation.
 83. The method of claim 78 wherein the oscillation isat a frequency sufficient to prevent settling of erythrocytes on asensor.
 84. The method of claim 78 further comprising the step of:storing an amperometric first sensor signal after a reagent isdissolved.
 85. The method of claim 78 further comprising the step of:analyzing subsequent amperometric sensor signals to determine themaximum rate of change in sensor signal.
 86. The method of claim 85further comprising the step of: determining a fixed fraction of themaximum rate of change in the sensor signal.
 87. The method of claim 85further comprising the step of: determining the coagulation parameterfrom the first sensor signal and the fixed fraction.
 88. A method ofassaying an enzyme in a sample of blood or blood derivative, comprisingthe steps of: introducing the sample into a cartridge which includes ananalysis location, metering a portion of the sample, moving the meteredsample to the analysis location, mixing the metered sample with reagentat the analysis location, allowing the enzyme to react with the reagent,positioning the reacted sample at a sensor, and detecting the product ofthe enzyme reaction using the sensor.
 89. A single-use cartridge used incombination with an analyzer for determining a coagulation parameter ofa sample of blood or blood derivative, comprising; a cartridge whichincludes an entry port for receiving a sample, an entry port closure, aholding chamber in communication at a first end with the entry port, anda capillary stop in communication with the holding chamber at a secondend, the capillary stop also in communication with an analysis chamber,the holding chamber in communication with an overflow chamber forreceiving and retaining excess sample, the overflow chamber incommunication with a pneumatic pump actuated by the analyzer, theanalyzer actuating the pneumatic pump to displace sample in the holdingchamber through the capillary stop into the analysis chamber to delivera metered portion of the sample into the analysis chamber, the analysischamber containing a substrate for the enzyme thrombin capable ofdissolving in the metered sample, an amperometric sensor for detectingthe product of the reaction between thrombin and the substrate, and aconductimetric sensor for detecting the position of the sample in theanalysis chamber, the amperometric sensor and the conductimetric sensorconnected to the analyzer for providing output signals to the analyzer,the analyzer capable of using the output signal of the conductimetricsensor to actuate the pneumatic pump to control the position of thesample in the analysis chamber, the analyzer capable of determining thecoagulation parameter from the output signal of the amperometric sensor,and the cartridge containing a hydrophobic area between the capillarystop and the analysis chamber to prevent sample in the analysis chamberfrom being drawn back into the holding chamber.